Ihc zinc fixative

Tissues collected from non-transplanted and P-Sp-NICD T cell transplanted mice were fixed in IHC Zinc Fixative (BD Pharmingen) and embedded in paraffin. Each organ paraffin block was serially sectioned at 5 μm and stained with Hematoxylin-Eosin. The slides were examined and scored blind by our pathologist.

Fixed-tumor tissues were excised from tumor-bearing mice and fixed using IHC Zinc Fixative (BD Bioscience, San Jose, CA, USA). Five micrometer paraffin-embedded sections were prepared for Immunohistochemical staining with Tyramide Signal Amplification (Opal Multiplex IHC Assay, PerkinElmer, Waltham, MA, USA) to examine the percentage of TILs. The following primary antibodies were used: rat anti-mouse CD3 (CD3-12), rabbit anti-mouse CD4 (EPR19514), rat anti-mouse CD8a (4SM15), rat anti-mouse Foxp3 (FJK-16S), rabbit anti-mouse PD1 (EPR20665). The number of immune cells per section was recorded blind to genotype and normalized to core area. Individual core counts from 10 or more replicates were available for most cases, and immune cell counts per square millimeter were averaged across replicates. Cut-off values of low versus high immune cells were defined by the midpoint. Slides were examined with an Olympus BX51 microscope using Camera Software for DP80 (Olympus America Inc., Center Valley, PA, USA). The cell count of TILs was determined using ImageJ. The density of TILs calculation as follows: $density of TIL=\frac{cells count number}{size of image field of view (m{m}^2)}$
$\begin{array}{ll}image field of& view \left(height, width, diagonal\right)\\ & =\frac{\mathrm{CCD}\mathrm{sensor}\mathrm{size}\left(\mathrm{height},\mathrm{width},\mathrm{diagonal}\right)}{\mathrm{objective}\mathrm{magnification}\mathrm{x}\mathrm{adapter}\mathrm{magnification}}\end{array}$

For histology, tumors were fixed in 4.5% formalin (SAV LP GmbH, Flintsbach am Inn, Germany) or zinc (IHC Zinc-Fixative, BD Biosciences, Franklin Lakes, USA) and embedded in paraffin (Paraplast® Embedding Media, McCormick Scientific, Leica Microsystems, Wetzlar, Germany). Formalin-fixed sections were cut at 4 μm, deparaffinized and stained with either H&E or with the respective antibodies for immunohistochemistry. Slides were stained using an automatic immunostainer (Discovery XT, Ventana Medical Systems, Inc., Tucson, USA) according to the manufacturer’s standard protocols with antibodies against CD31 (Abcam Inc., Cambridge, USA), β₃-Integrin (Abcam Inc.), glucose transporter 1 (GLUT1, Abcam Inc.) and Ki67 (ThermoFisher Scientific, Waltham, USA). Images were acquired under a microscope (Axio Imager A1, Carl Zeiss AG, Oberkochen, Germany) using a coupled digital camera (ProgRes® C10plus, JenOptik, Jena, Germany) and the software ImageAccess (Version 6, Imagic Bildverarbeitungs AG, Glattbrugg, Switzerland) or extracted from digitized whole slide imaging (Nanozoomer, Hamamatsu) using the corresponding software.

Mouse skin tissues were fixed with IHC zinc fixative (#550523; BD), processed to prepare paraffin blocks, sliced (3 µm thick), subjected to Elastica van Gieson staining, and observed with a microscope equipped with a digital camera (BX53/DP21; Olympus). For FBN staining, skin tissues submerged in Tissue‐Tek OCT Compound (Sakura Finetek) were rapidly frozen in liquid nitrogen. Cryosections (5 µm) placed on slide glasses were dried, fixed in cold acetone (−80°C) for 10 min, and incubated with primary antibodies, anti‐FBN1 (pAb‐9543: 1:150) or anti‐FBN2 (pAb868: 1:200; Charbonneau et al., 2003), for 3 hr at room temperature (RT). After washing with PBS without Ca²⁺ and Mg²⁺ (PBS), slices were stained with secondary antibodies (CF647 anti‐rabbit: 20282; 1:1,000; Biotium) for 30 min at RT, washed with PBS, and then nuclear counterstained with Hoechst 33342 (5 µg/ml) for 15 min. Images were recorded using a confocal microscope (TCS SP8; Leica). Transmission electron microscopy (TEM) was performed as described previously (Sakai & Keene, 1994).

Three days after the second delivery, three mice from each experimental group were sacrificed and tumors were excised and fixed in IHC zinc fixative (BD Pharmingen, BD Biosciences, San Jose, CA, USA) overnight and afterwards embedded in paraffin. Three consecutive 5-μm thick sections were cut from each paraffin block. The first section of each tumor sample was stained with hematoxylin and eosin (HE) for the determination of necrosis. The two remaining sections were immunohistochemically stained with rabbit monoclonal antibodies against Ki-67 (clone SP6, Thermo Fisher Scientific) at dilution 1:1200 for determination of proliferation of tumor cells or rabbit monoclonal antibodies against Cleaved Caspase-3 (Casp-3, Cell Signalling Technology, Danvers, MA, USA) at dilution 1:1500 for determination of apoptosis. As the colorogenic reagent a peroxidase-conjugated streptavidin–biotin system (Rabbit specific HRP/DAB detection IHC kit ab64261, Abcam, Cambridge, UK) was used followed by hematoxylin counterstaining. Three different fields of immunohistochemically stained sections were captured with a DP72 CCD camera (Olympus, Hamburg, Germany) connected to a BX-51 microscope (Olympus). Three independent observers blinded to the treatment groups quantified the percentage of tumor necrosis and the number of Ki-67- or Casp-3-positive cells on the acquired images.

Confluent NIH3T3 cells in 8-well chamber slides (8CS; SCS-008, Matsunami) were fixed with IHC Zinc Fixative (BD Biosciences) for 4.5 h and subjected to PLA using Duolink Green (Sigma-Aldrich) with anti-RECK (5B) and/or anti-ADAMTS10 (pAb-867) as primary antibodies and anti-Mouse PLUS and anti-Rabbit MINUS as secondary antibodies. PLA signals (green) and the number of nuclei (DAPI-stained) were quantified using ImageJ (NIH).